Normal cell development requires the interaction of RNA polymerase with many factors that regulate transcriptional activity. We study transcription from bacteriophage T4 middle promoters in which the initiation of transcription by E. coli RNA polymerase requires the phage-encoded transcriptional activator, MotA protein, and the T4 co-activator, AsiA protein. This system is a simple model for examining how factors can change the specificity of a polymerase. During infection, AsiA is found in a tight association with sigma70, the subunit of RNA polymerase that imparts specificity for promoter sequences. The presence of AsiA inhibits transcription from host and phage early promoters and is required, along with the T4 MotA protein, for activation of phage middle promoters. In the past year, we have investigated how AsiA gains access to the polymerase. Using single round transcription assays in vitro and native protein gel analyses, we have found that AsiA binds rapidly to free sigma70 at either 4o C or 30o C to form an AsiA-sigma70 complex that with polymerase core efficiently reconstitutes the AsiA-inhibited RNA polymerase holoenzyme. In contrast, AsiA does not inhibit transcription after a 15 min incubation with RNA polymerase holoenzyme at 4o C, and at 30o C an incubation of several minutes is required to inhibit most of the polymerase. We have found that the heat step that is required for AsiA inhibition is consistent with the momentary dissociation of holoenzyme to give free sigma70 and core. Thus, efficient generation of the AsiA-inhibited RNA polymerase requires that AsiA first binds to free sigma70 and then the AsiA-sigma complex binds to core to form the AsiA-inhibited polymerase. In vivo, free sigma70 is released from polymerase once polymerase has initiated transcription and begun to elongate the RNA. We suggest that this release provides AsiA with an opportunity to gain access to polymerase and that having free sigma70, rather than holoenzyme, as the target for AsiA helps to coordinate the start of middle transcription with the vitality of early promoter initiation.In RNA polymerase complexes with host promoters, two domains of sigma70 directly contact the DNA. An internal domain contacts the -10 region while a region within the C-terminal domain 4 contacts the -35 region. Previously, we have found that MotA is a transcriptional activator that binds to the -30 region of middle promoter DNA and like AsiA, forms a complex with sigma70. In the RNA polymerase/MotA/AsiA complex with middle promoter DNA, the protein-DNA contacts typical for host promoters are retained in the -10 region but upstream contacts are significantly altered. We have suggested that the interaction of MotA and AsiA with the C-terminal region of sigma70 is important to achieve this alteration. To test this hypothesis, we have collaborated with the laboratory of Ann Hochschild (Harvard Medical School) and adapted an E.coli two hybrid system that assays the interaction of a protein with domain 4 of sigma70 by the activity of a reporter lacZ gene. AsiA is known to bind tightly to domain 4 of sigma70 and we find, as expected, that there is a high level of lacZ activity when we test AsiA in this system. In addition, we observe a moderate level of lacZ activity when we test the N-terminal domain of MotA, which we have previously shown is needed for transcriptional activation. In contrast, the N-terminal domain of a MotA mutant, which we have shown is defective for transcriptional activation, gives a background level of lacZ activity in this system. We conclude that MotA, like AsiA, interacts with domain 4 of sigma70.